Better understanding of the serotonin neurotransmitter system and one of its key components, the serotonin transporter (SERT), is the primary aim of this project. The development of the SERT knockout mouse in the LCS provided a new living tool to study SERT and serotonin receptors, the target molecules for the largest numbers of neuropsychiatric drugs used in the world. 30-plus neurochemical, behavioral and other phenotypic changes have been discovered in the SERT knockout mouse. The resulting data is helping to guide the LCS and other laboratories in investigations of the multiple functional variants recently discovered in the human SERT gene.[unreadable] The broad goal of our studies is a better understanding of the serotonin neurotransmitter system and its contributions to physiology, behavior and human disorders, especially neuropsychiatric disorders. Serotonin has been implicated in almost every physiological function known. The serotonin transporter re-captures serotonin after its release, thereby terminating the action of serotonin at its receptors. [unreadable] In our attempt to better understand serotonin's function, we have generated a mouse model which either lacks the serotonin transporter or has a 50% reduction in serotonin transporter expression, produced by homologous recombination in embryonic stem cells. These SERT -/- and +/- mice have gene-proportionate increases in extracellular fluid serotonin (5-HT) concentrations. i.e., 9- or 5-fold excesses respectively over +/+ mice, with the SERT deficiency present since conception. At the same time, the SERT -/- mice have a 50% deficit of intracellular, releasable 5-HT. These mice also have significantly increased 5-HT synthesis and turnover across all five brain regions investigated in SERT -/- mice, and this increase is significantly greater in female SERT -/- mice as measured by HPLC-EC after decarboxylase inhibition (Kim et al., 2005). Dopamine and norepinephrine tissue concentrations and synthesis/turnover are unchanged. No changes in dopamine transporter sites, norepinephrine transporter sites or tryptophan hydroxylase or monoamine oxidase A/B enzyme activities or mRNA were observed (Kim et al., 2005). Dopaminergic neurons in substantia nigra accumulate excess serotonin in SERT -/- mice, and expression of organic cation transporters, OCT1 and OCT3, which are low affinity transporters of monoamines, is increased, indicative of some partial but inefficient attempts at compensation via heterologous transporters. SERT +/- mice with decreased SERT binding sites, decreased 5-HT clearance and elevated ECF 5-HT, nonetheless have unchanged tissue 5-HT concentrations in the brain and periphery and unchanged brain 5-HT synthesis and turnover. Thus, the loss of one SERT allele leads to a decrease in transporter function, but a single copy of SERT is adequate to maintain overall tissue 5-HT homeostasis (Kim et al., 2005).[unreadable] In models of anxiety, the SERT deficient mice exhibit markedly greater anxiety-like behaviors. Similarly, these mice have substantially greater activation of the HPA and sympathoadrenal systems in responses to minor stressful stimuli such as handling and saline injections, with high plasma ACTH and epinephrine increases, and more marked reductions in pituitary and adrenal gland hormones. The anxiety behaviors are restored to near normal levels by treatment with a serotonin 1A receptor antagonist or restoration of 5-HT1A function in the hypothalamus transgenic means. In a novel study completed this year, SERT-deficient female mice were found to be supersensitive to the mild stress response that follows exposure to a predator (Adamec et al., 2006).[unreadable] Exaggerated anxiety-like response (decreased center time and reduced activity in a brightly lit open field) were also observed this year by other collaborators in SERT-deficient mice created by C-terminal insertional mutagenesis (unlike the original mice constructed by N-terminus deletion of SERT exon 2 (Zhao et al., 2006).[unreadable] As serotonin has trophic effect on neuronal cells, we are investigating the role of the serotonin transporter and certain neurotrophic factors such as BDNF on neuronal function, survival, and development. We have successfully developed a SERT x BDNF double knockout mouse model to further investigate the role of the serotonin transporter and BDNF in the developing nervous system, as human genetic studies have separately implicated both genes in the development of affective and other neuropsychiatric disorders, including obsessive-compulsive disorder. In the past year, we reported that SERT -/- mice which lack one BDNF allele had more marked intracellular monoamine deficiencies and also had greater stress-response abnormalities and anxiety-related behaviors (Ren-Patterson et al., 2005). In a second report this year, we described how female SERT -/- x BDNF +/- mice were protected from these neurochemical and behavioral abnormalities, unlike male mice, and that this protection was lost after ovariectomy or male hormone treatments, i.e., revealing that epigenetic mechanisms are involved in this dual genetic phenotype (Ren-Patterson et al., 2006). Anatomical, cytoarchitectonic changes are found in the barrelfield layer IV cortex of SERT -/- and +/- mice, with a gene-dose-dependent reductions in barrel density observed using both CO and 5-HT autoradiography. To evaluate whether these cellular barrelfield changes were of functional importance, responses to whisker stimulation in the cortical barrel field were investigated this past year using local cerebral glucose utilization measurements with the 3H-2-deoxyglucose uptake technique (Esaki et al., 2005). In control, SERT +/+ mice, unilateral vibrissal stimulation led to an anticipated significant increase in glucose utilization in the contralateral somatosensory cortex relative to the unstimulated ipsilateral side. The magnitude of this response was significantly less in SERT -/- mice. In addition, reduced glucose utilization in response to vibrissal stimulation was also observed in the other components of the entire whisker pathway from the face to the cortex, including the ipsilateral spinal trigeminal nucleus, the sensory trigeminal nucleus, and the contralateral ventroposterior nucleus of the thalamus (Esaki et al., 2005). Rescue of the barrel architecture abnormalities in SERT -/- and +/-mice in the cortex was accomplished by treating these mice with the serotonin synthesis inhibitor, parachlorophenylalanine (PCPA) on postnatal days 1 and 2, but not later, indicating that these changes are fixed during the brief developmental time window. Marked but segmentally-incomplete genetic rescue also follows deletion of the 5-HT1B receptor, accomplished through interbreeding SERT -/- mice and 5-HT1B -/- mice ? indicating that excessive serotonergic signaling by the elevated ECF serotonin concentrations acting on the 5-HT1B receptors is responsible for the barrel field lesions.[unreadable] The SERT deficient mice manifest small to moderate changes in receptor numbers and, more variably, in their mRNA; however, these are accompanied by much greater changes in responses to selective receptor agonists. For example, hippocampal neuron firing rates, temperature and other responses to 8-OH-DPAT and ipsapirone (5-HT1A agonists), and locomotor and other responses to RU 24969, GR 127935 and CP 94253 (5-HT1B agonists) and the head and body twitch response to DOI (a 5-HT2A/2C agonist) are essentially absent in SERT -/- mice, with intermediate changes in SERT +/- mice on some measures. These diminished receptor-mediated responses are not attributable to changes in G proteins or G protein coupling, but our collaborative paper published last year reported markedly reduced signaling beyond the HTR2A receptors in the PLA2 arachidonic acid pathway in cortex (Qu et al., 2005).